CN103003444A

CN103003444A - Method and kit for the prognosis of colorectal cancer

Info

Publication number: CN103003444A
Application number: CN2010800672901A
Authority: CN
Inventors: 叶迅; 吴非; 徐清华; 刘芳; 孟夏; B·穆然
Original assignee: Biomerieux SA
Current assignee: Biomerieux SA
Priority date: 2010-06-08
Filing date: 2010-06-08
Publication date: 2013-03-27
Anticipated expiration: 2030-06-08
Also published as: WO2011153684A1; CN103003444B

Abstract

The present invention discloses a method and kit for the prognosis of colorectal cancer, especially a method comprising: a) obtaining the peripheral blood sample and extracting total RNA from the blood sample, b) contacting the total RNA with at least one reagent that is specific for at least one NK cell gene and no more than 25 specific reagents for 25 NK cell genes, c) determining the expression level of the at least one NK cell gene and of the most 25 NK cell genes to obtain an expression profile for the patient, and d) performing analysis of the expression profile of the patient.

Description

The method and the test kit that are used for the colorectal carcinoma prognosis

Invention field

The present invention relates to the colorectum cancer prognosis, be specifically related to method and test kit for this cancer prognosis.

Background technology

Colorectal carcinoma (CRC) is also referred to as colorectal carcinoma or large bowel cancer, is the 5th modal cancer form in the U.S., is the 4th common cancer and be the 3rd major cause that causes the cancer associated death in Europe in China.The early discovery of CRC remains the main challenge of publilc health.In fact, CRC is normally recoverable, when particularly the stage is diagnosed in early days.Some screening strategies have been taked at country variant.Conventional CRC examination test comprises fecal occult blood test (FOBT), sigmoidoscopy, colonoscopy, double-contrast barium examination or digital rectal examination.They all have advantage and limitation, but compliance still is lower than expection, mainly are because logistics (logistics) or patient's discomfort.

Over several years, seeking the blood marker that is used for early detection CRC becomes focus, particularly because its convenience.Simultaneously, the feasibility based on the test of blood has been supported in very small amount of research, and it shows that the gene biological marker in the blood can distinguish CRC patient and contrast.These researchs are based on flow cytometry, and it is for counting and check for example technology of cell (by they being suspended in liquid stream and making it pass through electronic detecting device) of microscopic particles.

The inventor finds that difference expression gene is mainly relevant with transportation with activated immune cell.Especially, they prove that natural killer cell (NK cell) represents biomarker important in the peripheral blood sample.They do not use classical Flow Cytometry, but determine difference expression gene in whole blood.Determine that by in whole blood, analyzing transcript gene expression dose is uncommon, because when those skilled in the art generally admitted in being diluted in complicated RNA mixture (total RNA), it was difficult obtaining customizing messages in the situation that does not have the specificity purification step.The advantage of present method is its step of also having avoided purifying RNA.

Therefore, the present invention relates to in the method for determining the colorectal carcinoma prognosis from patient's peripheral blood sample, described method comprises:

A) obtain described peripheral blood sample and extract total RNA from described blood sample,

B) described total RNA is contacted to the special reagent of 25 kinds of NK cytogenes with no more than 25 kinds to the special reagent of at least a NK cytogene with at least a,

C) determine described at least a NK cytogene and at the most the expression level of 25 kinds of NK cytogenes obtaining described patient's express spectra,

D) with before clinical classification for the patient's of good prognosis NK cellular gene expression and the NK cellular gene expression that is categorized as before the patient of poor prognosis described patient's express spectra is analyzed, wherein

If-described patient's express spectra with from the express spectra cluster (cluster) for the patient of poor prognosis of clinical classification before, determine that then described patient has poor prognosis, and

If-described patient's express spectra with from the express spectra cluster for the patient of good prognosis of clinical classification before, determine that then described patient has good prognosis.

Particularly, at above-mentioned steps b) in, total RNA is contacted the special reagent of 25 kinds of NK cytogenes with no more than 25 kinds the special reagent of at least a NK cytogene with at least a, described NK cytogene comprises the nucleotide sequence shown in the SEQ ID NO:1-12, and wherein said at least a reagent is special to being selected from following at least a NK cytogene:

(i) KLRB1 gene, it comprises for example full length sequence shown in the SEQ ID NO:1,

(ii) KLRC2 gene, it comprises for example full length sequence shown in the SEQ ID NO:2,3 or 4,

(iii) KLRC3 gene, it comprises for example full length sequence shown in the SEQ ID NO:5,6 or 7,

(iv) KLRD1 gene, it comprises for example full length sequence shown in the SEQ ID NO:8,9,10,11 or 12, and

(v) KLRK1 gene, it comprises for example full length sequence shown in the SEQ ID NO:13, and

At step c) in determine that the expression level of described at least a NK cytogene is to obtain patient's express spectra.

As describing in detail in experimental data, the expression level of at least a said gene is enough information to prediction CRC risk.

In one embodiment, at step b) in make described total RNA and the special reagent of at least 5 kinds of NK cytogenes and the combination of no more than 25 kinds of NK cytogenes contacted, wherein said reagent comprises the reagent special to following NK cytogene at least:

(v) KLRK1, it comprises for example full length sequence shown in the SEQ ID NO:13,

At step c) in determine that the expression level of described at least 4 kinds of NK cytogenes is to obtain patient's express spectra.

In addition, at step b) in, described total RNA is contacted with no more than 5 kinds of reagent to 5 kinds of target cell gene specifics with at least a reagent at least a target cell gene specific, described target cell gene comprises the nucleotide sequence shown in the SEQ ID NO:12-24, and wherein said at least a reagent is selected from following target cell gene specific at least a:

(i) GZMB gene, it comprises for example full length sequence shown in the SEQ ID NO:14,15,16 or 17,

(ii) CD247 gene, it comprises for example full length sequence shown in the SEQ ID NO:18,19 or 20,

(iii) RRAS2 gene, it comprises for example full length sequence shown in the SEQ ID NO:21 or 22, and

(iv) SH2D1B gene, it comprises for example full length sequence shown in the SEQ ID NO:23 or 24, and

(v) LCK gene, it comprises for example full length sequence shown in the SEQ ID NO:25,26,27,28,29 or 30, and

At step c) in determine that the expression level of described at least a target cell gene is to obtain patient's express spectra; And in one embodiment, make described total RNA and the special reagent of the combination of 5 kinds of target cell genes is contacted, wherein said reagent is to following target cell gene specific:

At step c) in determine that the expression level of described at least 5 kinds of cytogenes is to obtain patient's express spectra.

In another embodiment, at step b) in, described total RNA is contacted with no more than 100 kinds of reagent to 100 kinds of target cell gene specifics with at least a reagent at least a target cell gene specific, described target cell gene comprises the nucleotide sequence shown in the SEQ ID NO:25-59, and wherein said at least a reagent is to being selected from following at least a target cell gene specific:

(i) MRPS6 gene, it comprises for example full length sequence shown in the SEQ ID NO:31,32 or 33,

(ii) SPRY4 gene, it comprises for example full length sequence shown in the SEQ ID NO:34,

(iii) NEAT1 gene, it comprises for example full length sequence shown in the SEQ ID NO:35,

(iv) CYBB gene, it comprises for example full length sequence shown in the SEQ ID NO:36,

(v) DUSP2 gene, it comprises for example full length sequence shown in the SEQ ID NO:37,

(vi) PDEAD gene, it comprises for example full length sequence shown in the SEQ ID NO:38 or 39,

(vii) SH2D2A gene, it comprises for example full length sequence shown in the SEQ ID NO:40,41 or 42,

(viii) INSR gene, it comprises for example full length sequence shown in the SEQ ID NO:43 or 44,

(ix) ITGAM gene, it comprises for example full length sequence shown in the SEQ ID NO:45,

(x) VCAN gene, it comprises for example full length sequence shown in the SEQ ID NO:46,47,48 or 49,

(xi) CD163 gene, it comprises for example full length sequence shown in the SEQ ID NO:50 or 51,

(xii) P2RY10 gene, it comprises for example full length sequence shown in the SEQ ID NO:52 or 53,

(xii) CD226 gene, it comprises for example full length sequence shown in the SEQ ID NO:54,

(xiii) MRPL10 gene, it comprises for example full length sequence shown in the SEQ ID NO:55 or 56,

(xiv) ITPRIPL2 gene, it comprises for example full length sequence shown in the SEQ ID NO:57,

(xv) CD2 gene, it comprises for example full length sequence shown in the SEQ ID NO:58, and

(xvi) NUDT16 gene, it comprises for example full length sequence shown in the SEQ ID NO:59, and

At step c) in determine that the expression level of described at least a target cell gene is to obtain patient's express spectra.

Especially, at step b) in make described total RNA and the special reagent of at least 17 kinds of target cell genes and the combination of no more than 100 kinds of target cell genes contacted, wherein said reagent comprises the reagent to following target cell gene specific at least:

At step c) in determine that the expression level of described at least 17 kinds of target cell genes is to obtain patient's express spectra.

More properly, in the above-described method, step b) at least a specific reagent described in comprises at least a hybridization probe, comprises particularly at least a hybridization probe and at least a primer, and more specifically comprises at least a hybridization probe and two kinds of primers.

Total RNA comprise transfer RNA (tRNA), messenger RNA(mRNA) (mRNA) (for example from target gene transcribe and from the mRNA of any other genetic transcription) and ribosome-RNA(rRNA).

Be intended to explanation, the extraction of total RNA can realize with the step of the nucleic acid that discharges patient's cell and comprised by the cell that cracking is present in the blood sample.Be intended to use the cleavage method of describing as in patent application WO00/05338 (it is about mictomagnetism and mechanical lysis), WO 99/53304 (it is about electric cracking), WO99/15321 (it is about mechanical lysis) for example.Those skilled in the art can use other known cleavage method, for example heat-shocked or osmotic shock or use for example chemical cracking of guanidinesalt (U.S. Patent number 5,234,809) of chaotropic agent.Also may provide extra step, be used for other cellular constituent isolating nucleic acid from discharging in cleavage step.This makes condensed nucleic acid become possibility usually.Be intended to for example, can use randomly and (in this respect, see U.S. Patent number 4 by absorption or covalent effect with the coated magnetic-particle of oligonucleotide, 672,040 and U.S. Patent number 5,750,338), and can be incorporated into by the washing step purifying thus the nucleic acid of these magnetic-particles.If need to increase subsequently described nucleic acid, this nucleic acid purification step is particularly advantageous.The particularly advantageous embodiment of these magnetic-particles has been described in patent application WO-A-97/45202 and WO-A-99/35500.

Term " specific reagent " refers to such reagent, when it is contacted with biologic material as defined above, can with the special material of described target gene is combined.Be intended to explanation, when specific reagent and biologic material are the nuclear source, specific reagent contacts with biologic material can make specific reagent and the special material of described target gene is hybridized.Term " hybridization " refers to such process, namely during described process under suitable condition, thereby two nucleotide fragments form double-stranded mixture with stable and special hydrogen bonded.These hydrogen bonds between the VITAMIN B4 (A) of complementation and thymus pyrimidine (T) (or uridylic (U)) base (this is called as the A-T key), or between the guanine (G) of complementation and cytosine(Cyt) (C) base (this is called as the G-C key) form.The hybridization of two nucleotide fragments can be completely (being called complementary nucleotide fragment or sequence), and the double-stranded mixture that namely obtains in this crossover process only comprises A-T key and C-G key.This hybridization can be (being called abundant complementary nucleotide fragment or sequence) of part, and the double-stranded mixture that namely obtains had both comprised so that may form double-stranded A-T key and C-G key, also comprised the base of not being combined with complementary base.Article two, employed working conditions, particularly severity are depended in the hybridization between the nucleotide fragments.The severity specific definition be two nucleotide fragments based composition function and defined by the mispairing degree between two nucleotide fragments.Severity also depends on reaction parameter, for example is present in character and concentration and/or the hybridization temperature of ionic concn in the hybridization solution and type, denaturing agent.All these data are known, and can determine suitable condition by those skilled in the art.In general, the length of the nucleotide fragments of hybridizing as required, hybridization temperature is about 20 ℃ to 70 ℃, particularly 35 ℃ to 65 ℃, in the salts solution of the about 0.5-1M of concentration.Sequence, or nucleotide fragments, or oligonucleotide, or polynucleotide, be a series of Nucleotide motifs that fit together by the phosphoric acid ester bond, be characterized as the natural acid sequence that contains information, it can be hybridized with nucleotide fragments, described series may contain the monomer with different structure, and may obtain from the natural acid molecule and/or by genetic recombination and/or by chemosynthesis.Motif is the derivative of monomer, and described monomer can be the natural nucleotide of nucleic acid, and its composed component is sugar, phosphate group and nitrogenous base; In DNA, described sugar is deoxidation-2-ribose, and in RNA, described sugar is ribose; According to whether relating to DNA and RNA, nitrogenous base is selected from VITAMIN B4, guanine, uridylic, cytosine(Cyt) and thymus pyrimidine; Alternatively, monomer is at least a adorned Nucleotide in three kinds of composed components; Be intended to for example; modification can occur in the base level, and (base with modification is inosine for example; methyl-5-Deoxyribose cytidine; deoxyuridine; dimethylamino-5-FU; diamino-2; the 6-purine; the base of bromo-5-FU or any modification that other can be hybridized); or the level that occurs in sugar (replaces with the polymeric amide (people such as P.E.Nielsen such as at least one ribodesose; Science; 254; 1497-1500 (1991) [3])); or occur in addition the phosphate group level and (for example replace with ester, specifically be selected from bisphosphate; alkyl phosphate and acyl phosphate and thiophosphatephosphorothioate).

The specific embodiment according to the present invention, described specific reagent comprises at least a hybridization probe, or at least a hybridization probe and at least a primer special to target gene, or at least a hybridization probe and two kinds of primers special to target gene.

For the purposes of the present invention, term " amplimer " refers to nucleotide fragments, and it comprises 5-100 Nucleotide, preferred 15-30 Nucleotide, itself so that enzymatic polymerization for example enzymatic amplification react initial.Term " enzymatic amplification reaction " refers to produce by the effect of at least a enzyme the process of multiple copied nucleotide fragments.Such amplified reaction is well known to a person skilled in the art, and mention especially following technology: PCR (polymerase chain reaction) (as at U.S. Patent number 4,683,195, U.S. Patent number 4,683,202 and U.S. Patent number 4,800, describe in 159), LCR (ligase chain reaction (LCR)) (for example open in patent application EP 0,201 184), RCR (reparation chain reaction) (in patent application WO 90/01069, describing), 3SR (self-sustained sequence replication) (patent application WO 90/06995), NASBA (based on the amplification of nucleotide sequence) (patent application WO 91/02818), TMA (transcriptive intermediate amplification) (U.S. Patent number 5,399,491) and RT-PCR.

When enzymatic amplification was PCR, specific reagent comprised at least two kinds of amplimers that target gene is special, and it allows the material of amplified target gene specific.The special material of target gene is then preferably comprised by reverse transcription from the complementary DNA (being called the special cDNA of target gene) of the messenger RNA(mRNA) acquisition of target gene or the complementary RNA (being called the special cRNA of target gene) that obtains by transcribing the special cDNA of target gene.The PCR that finishes after enzymatic amplification is reverse transcription reaction is called RT-PCR.

Term " hybridization probe " refers to nucleotide fragments, it comprises at least 5 Nucleotide, 5-100 Nucleotide for example, 10-75 Nucleotide particularly, for example 15-35 Nucleotide and 60-70 Nucleotide are hybridized specificity and the special material of target gene are formed hybridization complex thereby it has under specified criteria.In the present invention, to the special material of target gene can be the nucleotide sequence (being called the special mRNA of target gene) that is included in the messenger RNA(mRNA) that is derived from target gene, be included in the complementary DNA that obtains by the described messenger RNA(mRNA) of reverse transcription nucleotide sequence (being called the special cDNA of target gene) or in addition for being included in by transcribing the nucleotide sequence (being called the special cRNA of target gene) in the complementary RNA that described cDNA described above obtains.Hybridization probe can comprise the label for its detection.Term " detection " refers to for example method of counting of direct-detection, or indirect detection, and it is by using the detection method of label.Exist many detection methods for detection of nucleic acid (see, for example, the people such as Kricka, ClinicalChemistry, 1999, no 45 (4), the 453-458 page or leaf, or the people such as Keller G.H., DNA Probes, 2nd Ed., Stockton Press, the 1993, the 5 and the 6th part, the 173-249 page or leaf).Term " label " refers to produce the tracer agent of the signal that can be detected.The nonrestrictive tabulation of these tracer agents comprises enzyme (signal that it can detect by for example colorimetric, fluorescence or luminous generation), for example horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes, glucose-6-phosphate dehydrogenase (G6PD); Chromophoric group, for example fluorescent agent, luminous agent or dye composition; The electron dense group, its can by Electron microscopy maybe can by they characteristic electron for example specific conductivity detect by Amperometric or voltammetry or by impedance measurement; Can change by optical means such as diffraction, surface plasma body resonant vibration or contact angle, or pass through for example atomic power spectrum of physics method, the group that tunnel effect etc. detect; Geigers, for example ³²P, ³⁵S or ¹²⁵I.

For the purposes of the present invention, hybridization probe can be " detection " probe.In this case, with label " detection " probe is carried out mark.Detection probes can be specially such as Tyagi and Kramer (Naturebiotech, 1996,14:303-308) described " molecular beacon " detection probes.These " molecular beacons " become fluorescent agent during hybridizing.They have stem-ring type structure and comprise fluorophore and " quencher " group.Special ring sequence and the combination of its complementary target nucleic acid sequence cause stem to launch and send fluorescent signal in suitable wavelength excitation processes.Detection probes can be specially " reporter probe " that comprise " barcode of colour coding (barecode) " according to the technology of NanoStringTM.

In order to detect hybridization, can use directly (specifically by label is mixed target sequence) or indirectly (specifically use detection probes as defined above) target sequence of mark.Especially, before hybridization step, may carry out the step of mark and/or cutting target sequence, for example the deoxyribonucleoside triphosphate of applying marking in the enzymatic amplification reaction process.Described cutting specifically can be finished by the effect of imidazoles or Manganous chloride tetrahydrate.Also can according to the sandwich hybridization technology of for example in document WO 91/19812, describing by with detection probe after amplification step the labels targets sequence.The preferred method of another concrete labeling nucleic acid has been described in application FR 2780059.

According to the preferred embodiment of the invention, detection probes comprises fluorophore and quencher.According to the present invention even preferred embodiment, hybridization probe comprises FAM (6-Fluoresceincarboxylic acid) or ROX (6-carboxyl-X-rhodamine) fluorophore and comprises quencher (Dabsyl) at its 3' end at its 5' end.

Hybridization probe also can be " catching " probe.In this case, by any appropriate means, namely directly or indirectly, for example by covalency or absorption, " catching " probe is fixed and maybe may be fixed on the solid substrate.As solid substrate, can use synthetic materials or natural materials (randomly as chemically modified), be specially polysaccharide for example based on cellulosic materials such as paper, derivatived cellulose is cellulose acetate and nitrocellulose or dextran for example, polymkeric substance, multipolymer (particularly styrene-based type monomer), natural fiber is cotton for example, and synthon nylon for example; Inorganic materials is silicon-dioxide, quartz, glass or pottery for example; Latex; Magnetic-particle; Metal derivative, gel etc.Solid substrate can be the form of microtiter plate, film (as describing) or particle in application WO-A-94/12670.Also some different capture probes may be fixed on the base material, every kind of capture probe is special to a kind of target gene.Particularly, can use biochip as base material, can fix a large amount of probes on it.Term " biochip " refers to the solid substrate of small volume, and a large amount of capture probes can connect thereon at preposition.The concept of biochip (or DNA chip) can be traced back to nineteen ninety for the initial stage.It has integrated microelectronics, nucleic acid chemistry, image analysis and information technology based on multidisciplinary technology.Principle of work is based on molecular biological basis: the hybridization phenomenon, namely two DNA and/or RNA sequence are by the pairing of base complementrity.Biochip method is based on the use that is connected to the capture probe on the solid substrate, and the target nucleotide fragment sample effect of mark fluorescent dyestuff is in described capture probe directly or indirectly.Capture probe is positioned on base material or the chip specifically, and each hybridization provides a specifying information relevant with the target nucleotide fragment.The information that obtains is cumulative, and for example making quantitatively, the expression level of one or more target gene becomes possibility.Then in order to analyze the expression of target gene, can prepare the base material that comprises a large amount of probes, described probe is all or part of corresponding to the target gene that is transcribed into mRNA.For the purposes of the present invention, term " low density substrate " refers to comprise the base material that is less than 50 kinds of probes.For the purposes of the present invention, term " middle density base material " refers to comprise the base material from 50 kinds of probe to 10000 kind of probes.For the purposes of the present invention, term " high-density base material " refers to comprise the base material more than 10000 kinds of probes.

CDNA or the cRNA that will be specific to subsequently the target gene of need analyzing are hybridized with for example special capture probe.After the hybridization, washing base material or chip, and cDNA or cRNA/ capture antibody mixture by the high-affinity part show tags of being combined with for example fluorescence dye type label.Read fluorescence and carry out fluorometric analysis by information technology with for example scanner.Be intended to explanation, can mention by Affymetrix company exploitation, be used for the DNA chip (" Accessing GeneticInformation with High-Density DNA arrays " of molecular diagnosis, the people such as M.Chee, Science, 1996,274,610-614." Light-generated oligonucleotide arrays for rapid DNAsequence analysis ", the people such as A.Caviani Pease, Proc.Natl.Acad.Sci.USA, 1994,91,5022-5026).In this technology, capture probe is generally less, about 25 Nucleotide.At publication G.Ramsay, Nature Biotechnology, 1998, No.16,40-44 page or leaf; F.Ginot, Human Mutation, 1997, No.10,1-10 page or leaf; The people such as J.Cheng, Molecular diagnosis, 1996, No.1 (3), 183-200 page or leaf; The people such as T.Livache, Nucleic Acids Research, 1994, No.22 (15), 2915-2921 page or leaf; The people such as J.Cheng, Nature Biotechnology, 1998, No.16,541-546 page or leaf or at U.S. Patent number 4,981,783, U.S. Patent number 5,700, and 637, U.S. Patent number 5,445,934, U.S. Patent number 5,744, and 305 and U.S. Patent number 5, provided other example of biochip in 807,522.The principal character of solid substrate should be and keeps capture probe to the hybridization characteristics of target nucleotide fragment, and produces simultaneously the background noise to the detection method minimum.Probe fixing on base material can be divided into three kinds of main Types.

At first, have the first technology, it is to deposit pre-synthesis probe.The connection of probe is finished by direct transfer, and it is by micropipet (micropipette) or little point (microdot) or pass through ink discharge device.This technology allows to connect magnitude range from the probe of several bases (5-10) to relatively large 60 bases (impact system) to hundreds of bases (microdeposit method).

Impact system is the change to the method for ink-jet printer use.It is based on advance very little liquid ball (volume＜1nl) with the speed that can reach 4000 drops/secs.Any contact between the system that impact system does not relate to releasing liquid and the liquid deposition surface thereon.

The microdeposit method is characterised in that the surface that tens of long probes to hundreds of bases is connected to glass slide.These probes usually extract from database and are through amplification and purified product form.The chip that this technology makes production be called microarray becomes possibility, and described chip carries about 10,000 DNA points at the surface-area (being called identified region) that is slightly less than 4 square centimeters.Yet, should not forget the nylon membrane purposes of (being called " VLA row (macroarray) "), its density with maximum 25 point/square centimeters is carried the product after the amplification (generally passing through PCR) of diameter 0.5mm to 1mm.This very flexibly technology is all used in many laboratories.In the present invention, biochip considers to comprise rear a kind of technology.Yet, in the situation such as patent application WO-A-00/71750 and FR 00/14896, can be with the sample deposition of the certain volume bottom in each hole of microtiter plate, or according to another patent application FR 00/14691, droplet deposition that can some amount is separated from one another is in same sterile petri dish bottom.

It is synthetic that the second is called original position for the technology that probe is connected in base material or chip.This technology causes directly producing short probe at chip surface.It is based on original position oligonucleotide synthetic (specifically seeing patent application WO 89/10977 and WO 90/03382) and based on the oligonucleotide synthesizer method.It is along glass surface mobile response chamber, and the Nucleotide extension occurs in described reaction chamber.

At last, the third technology is called optical lithography, and it is the method that is used for biochip of Affymetrix exploitation.It also is that original position is synthetic.Optical lithography comes from microprocessor technology.Modify chip surface by connecting photo-labile (can by photoactivation) chemical group.In case by illumination, these groups can react with the 3' end of oligonucleotide.By protecting this surface with the lightshade cover that limits shape, just optionally therefore illumination also activate the zone that expectation connects one of four kinds of Nucleotide or other Nucleotide.Continuously use different lightshade covers so that the protection/reaction cycle and so at about tens of square microns (μ m of may hocketing ²) point on produce oligonucleotide probe.This resolving power is so that may be at several square centimeters of (cm ²) surface-area on produce hundreds thousand of points of as many as.Optical lithography has advantage: parallel in batches, it is so that may only take advantage of N circulation to produce N aggressiveness chip through 4.All these technology can be used for the present invention.According to the preferred embodiment of the invention, step b as defined above) at least a specific reagent comprises at least a hybridization probe, and it preferably is fixed on the base material.This base material is preferably low, high or middle density base material as defined above.

In order to improve the amount of target genetic stocks, can before these hybridization steps that comprise on the base material of a large amount of probes, carry out enzymatic amplification reactions steps as defined above.

Can be by any scheme completing steps c well known by persons skilled in the art) the determining of the gene expression dose that hits.Generally speaking, can analyze by detecting the mRNA (messenger RNA(mRNA)) that transcribes from target gene at given time the expression of target gene.

The present invention preferably relates to by according to well known to a person skilled in the art that any scheme detection resources determines the expression level of target gene from the mRNA of target gene.The specific embodiment according to the present invention by detecting some different mRNA (every kind of mRNA is derived from a target gene), can be determined the expression level of some target genes simultaneously.

When specific reagent comprises at least a amplimer, may determine in the following manner the expression level of target gene: 1) extract total RNA (comprising transfer RNA (tRNA), ribosome-RNA(rRNA) (rRNA) and messenger RNA(mRNA) (mRNA)) afterwards from whole blood, carry out the complementary DNA (or cDNA) of reverse transcription step to obtain described mRNA.Be intended to explanation, can use and to carry out this reverse transcription reaction from the ThermoScript II of RNA fragment acquisition complementary DNA fragment.Specifically can use the ThermoScript II from AMV (avian myeloblastosis virus) or MMLV (Moroni murine leukemia virus).When more specifically only needing to obtain the cDNA of mRNA, in the situation that the nucleotide fragments that only comprises thymine alkali bases (poly T) exists, carry out this reverse transcription step, thereby described poly T is by forming poly T-poly A mixture with complementary hybridization of the poly A sequence of mRNA, and it is the initiation site of the reverse transcription reaction that undertaken by ThermoScript II of conduct then.Then obtain with the cDNA (cDNA that target gene is special) of the mRNA complementation that is derived from target gene and with the cDNA (cDNA that non-target gene is special) of the mRNA complementation that is derived from the gene outside the target gene.2) the special cDNA of the special amplimer of target gene and target gene is contacted with the special cDNA of non-target gene.The cDNA hybridization that the amplimer that target gene is special and target gene are special and the predetermined zone of the cDNA that increases specifically (it is derived from the mRNA from target gene) known length.The special cDNA of non-target gene is not amplified, thereby obtains the special cDNA of a large amount of target genes.For the purposes of the present invention, can indistinguishably quote " cDNA that target gene is special " or " being derived from the cDNA from the mRNA of target gene ".Can specifically carry out this step by the amplified reaction of PCR type or by the method for any other amplification technique as defined above.To PCR, also may be some to different amplimer (every a pair of primer is special to a target gene) the some different cDNA (each cDNA is special to different target genes) that increase simultaneously by using: be called multiplex amplification.3) by detect and quantitatively in above-mentioned steps 2) in the special cDNA of target gene of acquisition determine the expression of target gene.Can carry out this detection behind the big or small electrophoretic migration according to it at the special cDNA of target gene.The gel and the medium that are used for migration can comprise ethidium bromide, thereby after given transition time section, in the time of on gel being placed on UV (ultraviolet ray) platform, by the special cDNA of optical signal direct-detection target gene that sends.The amount of the cDNA that target gene is special is larger, and optical signal is just stronger.These electrophoretic techniques are known to those skilled in the art.Also can be with detecting the also cDNA of quantifying target gene specific by the quantitative scope that proceeds to saturated amplified reaction acquisition.For the difference of the enzyme efficient considering in different step (reverse transcription, PCR etc.), may observe, can be by determining simultaneously the not on the same group expression of patient's target gene of expression normalization method of " look after the house " gene (its expression is not similar among the patient on the same group).The ratio of expressing by obtaining expression of target gene and house-keeping gene, i.e. the ratio of the amount of the special cDNA of the amount by obtaining the special cDNA of target gene and house-keeping gene, any variation between the correction different experiments.Those skilled in the art are the following publication of reference specifically: Bustin S A, J Mol Endocrinol, 2002,29:23-39; Giulietti A Methods, 2001,25:386-401.

When specific reagent comprises at least a hybridization probe, may determine in the following manner the expression of target gene: after 1) extracting total RNA from whole blood, describe as mentioned ground and finish reverse transcription step, thus obtain and be derived from the mRNA complementation of target gene cDNA (cDNA that target gene is special) and with the cDNA (cDNA that non-target gene is special) of the mRNA complementation that is derived from the gene outside the target gene.2) all cDNA are contacted with base material, be fixed with the special capture probe of target gene of needs being analyzed its expression on the described base material, thereby carry out the special cDNA of target gene and the hybridization between the capture probe, but not the special cDNA of target gene is not hybridized with capture probe.Hybridization can comprise that all finish such as the solid substrate of the above-mentioned material of pointing out.According to the preferred embodiment of the invention, hybridization probe is fixed on the base material.Preferably, base material is low, high or middle density base material as defined above.Can before hybridization, be carried out by the step that the enzymatic amplification of the special cDNA of target gene as defined above forms, thereby obtain the special cDNA of a large amount of target genes and improve the special cDNA of target gene and possibility to the special capture probe hybridization of target gene.The step that also may carry out mark and/or cut the special cDNA of target gene described above before hybridization, for example the deoxyribonucleoside triphosphate of applying marking is used for amplified reaction.Described cutting specifically can be finished by the effect of imidazoles and Manganous chloride tetrahydrate.Also can be after the amplification step cDNA of labels targets gene specific, for example according to the sandwich hybridization technology of in document WO-A-91/19812, describing by with the probe hybridization of mark.The concrete grammar of other preferred mark and/or cutting nucleic acid has been described in application WO99/65926, WO 01/44507, WO 01/44506, WO 02/090584, WO 02/090319.3) carry out subsequently the step that forms by detecting hybridization.Described detection can contact with " detection " probe of mark by making base material (to the cDNA hybridization special with target gene thereon of the special capture probe of target gene), and the signal that detection is sent by label carries out.When the special cDNA of target gene has used label in advance, the signal that the direct-detection label sends.

When at step b) at least a specific reagent comprise at least a hybridization probe, also can determine in the following manner the expression of target gene: after 1) extracting total RNA from whole blood, finish as mentioned reverse transcription step with describing, thereby obtain the cDNA of the mRNA of biologic material.Use subsequently the T7 polysaccharase to carry out the polymerization of the complementary RNA of cDNA, described T7 polysaccharase is brought into play function also so that may obtain complementary RNA from dna profiling under promotor control.Obtain subsequently the cRNA of the cDNA of the special mRNA of the cRNA of cDNA of the special mRNA of target gene and non-target gene.2) all cRNA are contacted with base material, be fixed with the special capture probe of target gene of needs being analyzed its expression on the described base material, thereby carry out the special cRNA of target gene and the hybridization between the capture probe, but not the special cRNA of target gene is not hybridized with capture probe.When needs are analyzed the expression of some target genes simultaneously, can fix some different capture probes at base material, each probe is special to a target gene.The step that also may before hybridization, carry out mark and/or cut the special cRNA of target gene described above.3) carry out subsequently the step that forms by detecting hybridization.Detection can contact with " detection " probe that is marked with label by making base material (to the cRNA hybridization special with target gene thereon of the special capture probe of target gene), and detection is finished by the signal that label sends.When the special cRNA of target gene has used label in advance, the signal that the direct-detection label sends.When hybridization had the biochip class base material of a large amount of probes on using it, it was particularly advantageous using cRNA.

The present invention also comprises test kit, it is used at the peripheral blood sample prognosis colorectal carcinoma from the patient, described test kit comprise at least a to the special reagent of at least a NK cytogene and no more than 25 kinds to 25 kinds of reagent that the NK cytogene is special, described NK cytogene comprises the nucleotide sequence shown in the SEQ ID NO:1-13 at least, and to be selected from following NK cytogene special at least a for wherein said at least a reagent:

(v) KLRK1 gene, it comprises for example full length sequence shown in the SEQ ID NO:13.

In one embodiment, test kit comprises the combination to the special reagent of following NK cytogene:

In such embodiments, but the some NK cytogenes of specific reagent target but the combination of no more than 25 NK genes.

In addition, test kit can comprise at least a reagent at least a target cell gene and no more than 5 kinds of target cell gene specifics, and described at least a target cell gene is selected from:

(v) LCK gene, it comprises for example full length sequence shown in the SEQ ID NO:25,26,27,28,29 or 30.

Particularly, it comprises 5 kinds of reagent to following target cell gene specific:

In such embodiments, but for example above-described some target cell genes but the combination of no more than 5 kinds of target cell genes of specific reagent target.

In another embodiment, routine test kit as defined above can comprise at least a to the reagent of at least a target cell gene specific and 100 kinds of reagent to 100 kinds of target cell gene specifics at the most, and described at least a target cell gene is selected from:

(xvi) NUDT16 gene, it comprises for example full length sequence shown in the SEQ ID NO:59.

And particularly, it comprises 17 kinds of reagent to following 17 kinds of target cell gene specifics:

In such embodiments, but for example above-described some target cell genes but the combination of no more than 100 kinds of target cell genes of specific reagent target.

As explained above, described at least a specific reagent comprises at least a hybridization probe, specifically comprises at least a hybridization probe and at least a primer, and more specifically comprises at least a hybridization probe and two kinds of primers.

At last, the present invention relates at least a to the special reagent of at least a NK cytogene and no more than 25 kinds to 25 kinds of reagent purposes in the preparation composition that the NK cytogene is special, described composition is used at the biological sample prognosis colorectal carcinoma from the patient, described NK cytogene comprises the nucleotide sequence shown in the SEQ ID NO:1-12, and wherein said at least a reagent is special to comprising at least a NK cytogene that is selected from the nucleotide sequence of SEQ ID NO:1-12 shown in arbitrary;

Particularly to the purposes of the special reagent of the combination of at least 5 kinds of NK cytogenes and no more than 25 kinds of NK cytogenes in the preparation composition, described composition is used at the biological sample prognosis colorectal carcinoma from the patient, and wherein said reagent is special to comprising at least 5 kinds of NK cytogenes that are selected from respectively the nucleotide sequence shown in SEQ ID NO:1,2-4,5-7 and the 8-12;

Particularly to the purposes of the special reagent of the combination of 10 kinds of target cell genes in the preparation composition, described composition is used at the biological sample prognosis colorectal carcinoma from the patient, and wherein said reagent is special to comprising the target cell gene that is selected from respectively SEQ ID NO:1,2-4,5-7,8-12,13,14-17,18-20,21-22,23-24 and the described nucleotide sequence of 25-30; With

More particularly special to the combination of 10 kinds of target cell genes and the no more than 100 kinds of target genes purposes of reagent in the preparation composition, described composition is used at the biological sample prognosis colorectal carcinoma from the patient, and wherein said reagent is selected from respectively SEQ ID NO:1 to comprising, 2-4,5-7,8-12,13,14-17,18-20,21-22,23-24,25-30,31-33,34,35,36,37,38-39,4-42,43-44,45,46-49,50-51,52-53,54,55-56,57, the target cell gene of the nucleotide sequence shown in 58 and 59 is special;

Wherein, described at least a specific reagent comprises at least a hybridization probe, at least a hybridization probe and at least a primer or at least a hybridization probe and two kinds of primers.

Summary of drawings

NK cell score in colonoscopy negative control (CNC) and colorectal carcinoma (CRC) patient's blood sample, and the CRC sample is according to the distribution of carcinoma stage.The circular CNC that represents; Square frame, positive triangle, inverted triangle and rhombus represent respectively I phase, II phase, III phase and IV phase CRC.

Embodiment

I) materials and methods

1. patient and sample collection

This research has obtained the approval of local clinical study Ethics Committee.Obtained all participants' written Informed Consent Form.

To the CRC group, between in July, 2006 and in March, 2008,119 colorectal cancer patients have been recruited continuously at tumour hospital of Chinese Fudan University (FUCH) colorectal surgery for this research.Tumour-lymphoglandula of recommending according to International Union Against Cancer (UICC)-transfer system is to neoplasm staging.There is not the patient to accept preoperation radiotherapy or chemotherapy.The patient who suffers from heredity colorectal carcinoma or inflammatory bowel disease (Crohn disease or ulcerative colitis) has been got rid of in this research.To each patient, after at least one week, before the operation, the 2.5ml peripheral blood is collected into PAXgene at colonoscopy ^TMIn the blood rna pipe (PreAnalytiX GmbH, Hombrechtikon, CH), and process according to manufacturer specification.To control group, register 101 from the community hospital of District of Shanghai and confirmed that by colonoscopy the FOBT that does not carry any polyp or colorectum cancerous symptom tests positive participant.Be collected into peripheral blood sample in the PAXgene pipe the last week in the colonoscopy inspection.All participants' that this research comprises detailed features provides in table 1.

Table 1: patient characteristic

2.RNA extract and Microarray Experiments

Follow manufacturer specification PAXgene ^TMBlood rna system (PreAnalytix) extracts total RNA.By the amount of spectrophotometer at the total RNA of photo densitometry of 260 nanometers, at the upper RNA 6000Nano that uses of BioAnalyzer Agilent 2100 (Agilent Technologies, PaloAlto, CA, U.S.A.)

The test kit quality of evaluation.Only analyze the sample that has 7 to 10 RNA integrity numerical value.Then use Ribo-SPIA with the total RNA reverse transcription of 50 nanograms and according to the manufacturing mark quasi-project ^TMThe WT-Ovation of technology (NuGEN Technologies Inc., San Carlos, CA, U.S.A.) ^TMRNA amplification system linear amplification is strand cDNA, and uses QIAquick ^TMPCR purification kit (QIAGEN GmbH, Hilden, Germany) purified product.Use subsequently RQ1 without DNA enzyme (the Promega Corp. of RNA enzyme, Fitchburg, WI, U.S.A.) cDNA behind 2 milligrams of amplifications of fragmentation and the purifying, and with biotinylated deoxynucleoside triphosphate by terminal enzyme (DNA) (Roche Diagnostics Corp., Indianapoli, IN, U.S.A.) and Dna marker reagent (Affymetrix Inc., Santa Clara, CA, U.S.A) mark.In hybrid heater 640 (AgilentTechnologies) 50 ℃ 18 minutes, per minute 60 turns hybridizes the cDNA of mark to HG U133Plus 2.0Array (Affymetrix).HG U133Plus 2.0Array comprises 54,675 probe sets, and it has represented about 39,000 people's genes that characterize best.After the hybridization, use according to Affymetrix scheme EukGE-WS2v4 Fluidics Station 450 (Affymetrix) washing microarray and dyeing.With

Scanner 3000 (Affymetrix) scans microarray.

3. microarray data analysis

Affymetrix quality-controlling parameters suggestion difficulty action accomplishment control analysis according to standard.Based on evaluation criteria, we have reached minimum quality requirement at all experiments.Smoothly conclude pre-treatment Affymetrix by RMA (the robust multi-chip is average) with background correction, fractile normalization method and median and express microarray [1].To have extreme strength of signal and (be lower than 50 or be higher than 2 * 10 ¹⁴) probe sets filter.In order to reduce the possibility of batch effect, the expression data after filtering is used normalization algorithm CamBat ¹¹CamBat method (http://statistics.byu.edu/johnson/ComBat/) application parameter or distribution free experience Bei Shi framework are concentrated at data-oriented and are regulated batch effect.Equal 0.05 time definite difference expression gene (DEG) by microarray significance analysis (SAM) at false discovery rate (FDR) ¹²Use has the R environment in Bioconductor storehouse and carries out pre-treatment and statistics step.Use Ingenuity Pathway Analysis software 8.5 versions (Ingenuity Systems, Redwood City, CA, U.S.A) to carry out gene ontology (Gene Ontology) and classical pathway (Canonical Pathway) analysis.

II) result

1. colorectal carcinoma and control patients group's feature

Clinical and the demography variable of 119 colorectal carcinoma (CRC) patients and 101 colonoscopy negative controls (CNC) is summarized in table 1.To CRC, behind colonoscopy, confirmed the diagnosis of colorectal carcinoma by the pathologist.From select contrast the FOBT positive patient of community hospital's registration, they are finally negative at the colonoscopy that tumour hospital of Fudan University (FUCH) carries out.Between CRC and CNC group well balance age and sex.

2. the evaluation of the different gene in colorectal cancer patients and colonoscopy negative control of the expression in the peripheral blood

The contriver seeks the difference expression gene (DEG) of the High Defferential of between two groups tool in 119 CRC and 101 CNC, CRC group (I, II, III and IV phase) is considered as a whole.After suitable pre-treatment, kept 20169 probe sets and carried out the DEG analysis.Equal 0.05 at FDR, multiple changes (FC) greater than 1.2, uses SAM to identify 327 DEG.

In these 327 DEG, find respectively 195 (59.6%) and 132 (40.36%) in the CRC sample with higher and low expression level more.The P value scope of T check is from 1.43 * 10 ^-25To 1.51 * 10 ^-01, 18 DEG have less than 6.27 * 10 ^-15T check P value and all correspondence the gene of fine note: MRPS6, SPRY4, NEAT1, CYBB, DUSP2, PDE4D, SH2D2A, G (1-2) NSR, ITGAM, VCAN, CD163, P2RY10, CD226, MRPL10, ITPRIPL2, CD2 and NUDT16 (table 2).It is 1.83 (the higher levels of NEAT1 of tool among the CRC) and 1.71 (the lower level HBG2 of tool among the CRC) that the highest multiple changes (FC) value, and 26 (8%) among 327 DEG have and are higher than 1.40 FC value.

For example, to SPRY4 (among the CRC more high expression level rank the first T check P value 4.04 * 10 ^-23, FC 1.79) and MRPS6 (lower expression level ranks the first among the CRC, T check P value 1.43 * 10 ^-25, FC 1.27) and the result that observes.Such example has represented the gene that significant difference is expressed between CRC and CNC patient.To SPRY4,101 CNC have observed very similarly hybridization signal value, and the value of CRC is more various but compare with CNC and to have significantly (p value 4.04 * 10 ^-23) mean value (FC 1.78) that improves.To MRPS6, two colonies have all shown similar dispersiveness, and the CRC tool is (p value 1.43 * 10 significantly ^-25) mean value (FC 1.27) that reduces.

In front 18 DEG, observe 4 film leukocyte marker things, expression levels different in CRC patient's peripheral blood is compared in demonstration from CNC: lower level CD2 and CD226, it is respectively by the T cell with mainly by the NK cell expressing; Higher levels of CD163 and CD11B (ITGAM), it is mainly expressed by monocyte and many white corpuscles that relates to innate immune system respectively.What is interesting is equally in cytotoxic T lymphocyte and NK cell (NK) cell the granzyme B by the GZMB genes encoding, the lower expression in the CRC sample.Other gene is reported as the part of different signal paths as INSR, SPRY4, DUSP2, PDE4D and ITPRIPL2, and SH2D2A is reported as the T cell-specific.Reported that VCAN expresses in monocyte, and its expression level higher in the CRC sample and CD163 and ITGAM together can compare certain activation of the circulating monocytic cell in peripheral blood with CNC relevant with these patients.

By using Ingenuity Pathway Analysis (IPA) that 327 DEG are analyzed, it returns the ID of 321 location, and it is suitable for explaining relevant biological function (Bio Fuctions) and classical pathway.Grow and function (Physiological SystemDevelopmentand Function) for the physiology system, (the p value is from 1.44 * 10 for the tool high score to observe immunocyte transportation (Immune Cell Trafficking) ^-12To 1.57 * 10 ^-02, comprise 50 kinds of molecules), it has been contained activation, migration, accumulation, interior stream, chemotaxis, cellular invasion, cell movement, chemical induction (chemoattraction), pre-sharp (priming) of panimmunity cell and has adhered to.What is interesting is that for classical pathway natural killer cell signal transduction (Natural Killer Cell Signaling) is to have minimum P value (2.55 * 10 ^-05) one, it comprises 10 gene: CD247, KLRB1, KLRC2, KLRC3, KLRD1, KLRK1, LCK, PRKCH, RRAS2 and SH2D1D.The relating to of the membrane receptor of 5 NK cell-specifics (KLRB1, KLRC2, KLRC3, KLRD1, KLRK1), show in CRC patient's peripheral blood the specific NK cellular component difference at gene expression dose very consumingly.NK cellular gene expressions all in CRC reduce.The result is summarized in following table 2 and 3.

Table 2: front 18 genes (DEG) of expressing at contrast (CNC) the patient sample difference of colorectal carcinoma (CRC) and colonoscopy feminine gender; Gene is described, and T check P value changes relevant information with multiple

* from NetAffx ^TMWith from Ingenuity Pathway

8.5 the gene of version is described

Table 3:NK cell score: the gene of selection, T check P value and multiple change related information

* from NetAffx ^TMWith from Ingenuity Pathway

8.5 the gene of version is described

The gene relevant to these 10 NK cells observed lower expression level in the CRC group, and prompting circulation NK cell number reduces, or such cell outflows to other organ-/ tissue compartment especially knub position.It also is noticeable observing lower level GZMB expression, and representative occurs in the main matter of cytotoxicity level in CRC patient.

Contact (Communication betweenInnate and Adaptive Immune Cells) and the iCOS-iCOSL signal in the t helper cell that the most front classical pathway of rank relates between φt cell receptor signal conduction (T Cell ReceptorSignaling), the congenital and adaptive immunity cell conduct (iCOS-iCOSL Signaling in T Helper Cells), and it has respectively 9.08 * 10 ^-05, 2.85 * 10 ^-04With 5.78 * 10 ^-04The P value.

What is interesting is, to 51 in 119 CRC patients' the sample, observe and be lower than front 1/4th low NK cell score, and in 101 CNC patient's samples only 4.Use so direct cutoff value, the performance of this differentiation can be expressed as 43% sensitivity and 96% specificity.In addition, when distinguishing CRC patient according to the tumour TNM phase (I phase, II phase, III phase or IV phase), we observe this NK cell score and reduce gradually (Fig. 1) from the I phase to the IV phase in CRC patients.Mainly between CNC and II phase, III phase and IV phase CRC, and between I phase CRC and II-III and IV phase CRC, observe the difference of statistically significant.

This studies show that transcribes the potentiality that group is found biological marker in the peripheral blood, and provides new understanding to the immunne response in the colorectal carcinoma.Except existing filtering mode being prepared possible substituting/complementation, these results have also shown the expression analysis of for example relevant with NK cell gene so that can distinguish the patient who suffers from colorectal carcinoma, thereby have opened the gate of personalized medicine.

Reference

1.Irizarry?RA,Hobbs?B,Collin?F,Beazer-Barclay?YD,Antonellis?KJ,Scherf?U,Speed?TP.Exploration,normalization,and?summaries?of?high?densityoligonucleotide?array?probe?level?data.Biostatistics?2003;4:249-64)

2.Johnson?WE,Li?C,Rabinovic?A.Adjusting?batch?effects?in?microarrayexpression?data?using?empirical?Bayes?methods.Biostatistics?2007;8:118-27.

3.Tusher?VG，Tibshirani?R,Chu?G.Significance?analysis?of?microarraysapplied?to?the?ionizing?radiation?response.Proc?Natl?Acad?Sci?USA2001;98:5116-21.

4.Team?RDC.R:A?Language?and?Environment?for?Statistical?Computing.Vienna,Austria,2009.

Claims

1. method, it is used for determining the colorectal carcinoma prognosis in the peripheral blood sample from the patient that described method comprises:

A) obtain described peripheral blood sample and from described blood sample, extract total RNA,

If-described patient's express spectra with from the express spectra cluster for the patient of poor prognosis of clinical classification before, determine that then described patient has poor prognosis, and

2. the method for claim 1, wherein at step b) in described total RNA is contacted the special reagent of 25 kinds of NK cytogenes with no more than 25 kinds the special reagent of at least a NK cytogene with at least a, described NK cytogene comprises the nucleotide sequence shown in the SEQ ID NO:1-12, and wherein said at least a reagent is special to being selected from following at least a NK cytogene:

At step c) in determine that the expression level of described at least a NK cytogene is to obtain described patient's express spectra.

3. the method for claim 2 is wherein at step b) in make described total RNA and the special reagent of at least 5 kinds of NK cytogenes and the combination of no more than 25 kinds of NK cytogenes contacted, wherein said reagent comprises at least to the special reagent of following NK cytogene:

4. the method for claim 3, wherein at step b) in described total RNA is contacted with no more than 5 kinds of reagent to 5 kinds of target cell gene specifics with at least a reagent at least a target cell gene specific, described target cell gene comprises the nucleotide sequence shown in the SEQ ID NO:12-24, and wherein said at least a reagent is to being selected from following at least a target cell gene specific:

At step c) in determine that the expression level of described at least a target cell gene is to obtain described patient's express spectra.

5. the method for claim 4 is wherein at step b) in make described total RNA and the special reagent of the combination of 5 kinds of target cell genes contacted, wherein said reagent is to following target cell gene specific:

At step c) in determine that the expression level of described at least 5 kinds of cytogenes is to obtain described patient's express spectra.

6. the method for claim 5, wherein at step b) in described total RNA is contacted with no more than 100 kinds of reagent to 100 kinds of target cell gene specifics with at least a reagent at least a target cell gene specific, described target cell gene comprises the nucleotide sequence shown in the SEQ ID NO:25-59, and wherein said at least a reagent is to being selected from following at least a target cell gene specific:

7. the method for claim 6 is wherein at rapid b) in make described total RNA and the special reagent of at least 17 kinds of target cell genes and the combination of no more than 100 kinds of target cell genes contacted, wherein said reagent comprises the reagent to following target cell gene specific at least:

At step c) in determine that the expression level of described at least 17 kinds of target cell genes is to obtain described patient's express spectra.

8. each method, wherein step b in the aforementioned claim) described at least a specific reagent comprise at least a hybridization probe.

9. the method for claim 8, wherein step b) described in specific reagent comprise at least a hybridization probe and at least a primer.

10. the method for claim 7, wherein step b) described in specific reagent comprise at least a hybridization probe and two kinds of primers.

11. test kit, it is used at the peripheral blood sample prognosis colorectal carcinoma from the patient, described test kit comprise at least a to the special reagent of at least a NK cytogene and no more than 25 kinds to 25 kinds of reagent that the NK cytogene is special, described NK cytogene comprises the nucleotide sequence shown in the SEQ IDNO:1-13 at least, and to be selected from following NK cytogene special at least a for wherein said at least a reagent:

12. the test kit of claim 11, it comprises the special reagent of following NK cytogene:

13. the test kit of claim 12, it comprises at least a reagent at least a target cell gene and no more than 5 kinds of target cell gene specifics, and described at least a target cell gene is selected from:

14. the test kit of claim 13, it comprises 5 kinds of reagent to following target cell gene specific:

15. the test kit of claim 14, it comprises at least a to the reagent of at least a target cell gene specific and 100 kinds of reagent to 100 kinds of target cell gene specifics at the most, and described target cell gene at least is selected from:

16. the test kit of claim 15, it comprises 17 kinds of reagent to following 17 kinds of target cell gene specifics:

17. at least a to the special reagent of at least a NK cytogene and no more than 25 kinds to 25 kinds of reagent purposes in the preparation composition that the NK cytogene is special, described composition is used at the biological sample prognosis colorectal carcinoma from the patient, described NK cytogene comprises the nucleotide sequence shown in the SEQ IDNO:1-12, and wherein said at least a reagent is special to comprising at least a NK cytogene that is selected from the nucleotide sequence shown in arbitrary among the SEQ IDNO:1-12.

18. reagent the purposes in preparation composition special to the combination of at least 5 kinds of NK cytogenes and no more than 25 kinds of NK cytogenes, described composition is used at the biological sample prognosis colorectal carcinoma from the patient, and wherein said reagent is special to comprising at least 5 kinds of NK cytogenes that are selected from respectively the nucleotide sequence shown in SEQ ID NO:1,2-4,5-7 and the 8-12.

19. reagent the purposes in preparation composition special to the combination of 10 kinds of target cell genes, described composition is used at the biological sample prognosis colorectal carcinoma from the patient, and wherein said reagent is special to comprising the target cell gene that is selected from respectively the nucleotide sequence shown in SEQ ID NO:1,2-4,5-7,8-12,13,14-17,18-20,21-22,23-24 and the 25-30.

20. reagent the purposes in preparation composition special to the combination of 10 kinds of target cell genes and no more than 100 kinds of target genes, described composition is used at the biological sample prognosis colorectal carcinoma from the patient, and wherein said reagent is special to comprising the target cell gene that is selected from respectively the nucleotide sequence shown in SEQ ID NO:1,2-4,5-7,8-12,13,14-17,18-20,21-22,23-24,25-30,31-33,34,35,36,37,38-39,4-42,43-44,45,46-49,50-51,52-53,54, the 55-56,57,58 and 59.

21. each purposes among the claim 17-20, wherein said at least a specific reagent comprises at least a hybridization probe.

22. each purposes among the claim 17-20, wherein said at least a specific reagent comprises at least a hybridization probe and at least a primer.

23. each purposes among the claim 17-20, wherein said at least a specific reagent comprise at least a hybridization probe and two kinds of primers.